Physiological processes which, firstly, ensure the fluidity of the blood in the vascular system and, secondly, make sure extravascular blood loss is avoided through the formation of blood clots come under the term hemostasis. The regulation of hemostasis involves a multiplicity of protein factors and also cellular components, for example thrombocytes (platelets). In the event of vascular injury, there is initially attachment of thrombocytes to the subendothelial collagen. This adhesion is mediated by adhesive proteins, such as von Willebrand factor (VWF). During the adhesion process, the thrombocytes are activated and release mediators from their granules, inducing the aggregation of further thrombocytes and intensification of activation. This achieves primary vascular wall occlusion (primary hemostasis), which needs further reactions of the plasmatic coagulation system (secondary hemostasis) to stabilize it. Dysregulation of these processes may lead to thrombophilia or bleeding diathesis and, depending on the severity, life-threatening sequelae.
In coagulation diagnostics, various methods and systems are known which make it possible to determine whether the blood of a patient can coagulate properly or whether a coagulation defect is present. In the event of a coagulation defect, it is often necessary to obtain more precise information about the cause of the defect present in order to be able to select optimal therapeutic measures. An important subfunction of the coagulation system which can be tested specifically is primary hemostasis, which depends substantially on the functional efficiency of thrombocytes.
One known method for testing thrombocyte function is that of bleeding time determination. This is an in vivo global test which captures primary hemostasis. The bleeding time is determined by causing the patient a small cutting or stabbing injury and measuring the time for bleeding to stop. This is a rough, difficult-to-standardize test which is used especially in emergency situations to obtain a snapshot of primary hemostasis. The intake of thrombocyte aggregation inhibitors leads to prolongation of bleeding time. A disadvantage of bleeding time determination is that even if bleeding time is normal, a thrombocyte function defect cannot be ruled out.
In vitro methods permit substantially more sensitive detection of thrombocyte function defects. Typically, in these methods, aggregation of thrombocytes is induced in a whole blood sample or in a sample of platelet-rich plasma (PRP) by addition of an activator and the aggregation reaction is measured. The most commonly used activators to induce thrombocyte aggregation are: ADP (adenosine 5′-diphosphate), collagen, epinephrine (adrenaline), ristocetin and various combinations thereof and thrombin, TRAP (thrombin receptor activating protein), U-46619, heparin (especially in the case of heparin-induced thrombocytopenia) or serotonin.
One known system for determining thrombocyte function in vitro is what is known as the Platelet Function Analyzer system, or PFA system for short (PFA-100®, PFA-200, Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). Using the PFA system, primary hemostasis is measured in whole blood samples under flow conditions and hence in the presence of high shear forces.
To simulate the flow conditions and the shear forces which prevail minor arterial blood vessels, a negative pressure of about −40 mbar is generated in a PFA measuring cell inserted into a PFA analysis device, and the citrated whole blood, which is located in a sample reservoir, flows through a capillary which has a diameter of about 200 μm. The capillary opens into a measuring chamber which is closed off by a partition element, for example a membrane, which contains a central capillary aperture through which the blood flows owing to the negative pressure. In most cases, the membrane, at least in the region around the aperture, contains one or more activators which induce thrombocyte aggregation, and so the blood which flows past comes into contact with the aggregation-inducing substances in the region of the aperture. The induced adhesion and aggregation of the thrombocytes results, in the region of the aperture, in the formation of a platelet plug (thrombus) which closes the membrane aperture and stops the blood flow. In this system, the time to closure of the membrane aperture is measured. This “closure time” correlates with the functional efficiency of the thrombocytes. A measuring cell for use in a method for determining thrombocyte function on the basis of closure time is, for example, described in the patent document WO 97/34698. To date, the method for determining closure time uses measuring cells which have a membrane coated with collagen (Col) and additionally either ADP or epinephrine (Epi). Other measuring cells which are especially suitable for determining antithrombotics from the group of the P2Y(12) antagonists, for example clopidogrel, have a membrane which contains ADP and prostaglandin E1 (INNOVANCE® PFA P2Y, EP-A1-1850134).
One known method for determining thrombocyte function in a sample using a PFA system comprises the following steps:                a) using a measuring cell, wherein the measuring cell comprises the following elements:        
a retention chamber for retaining the sample,                b) a capillary through which the sample is conducted from the retention chamber into a measuring chamber,        c) a measuring chamber which is divided by a partition element into two compartments, wherein the first compartment accommodates the sample from the capillary,        d) a partition element which divides the measuring chamber into two compartments and which has an aperture through which the sample can flow from the first compartment into the second compartment;        e) filling the retention chamber of the measuring cell with the sample;        f) placing the measuring cell in a device for automatic determination of thrombocyte function, wherein the device comprises the following elements:        
means for applying negative pressure in the measuring chamber of the measuring cell,                g) means for determining the total volume which, as a result of the application of negative pressure, is drawn from the measuring cell when negative pressure is applied in the measuring chamber of the measuring cell;        h) applying negative pressure in the measuring chamber of the measuring cell and conducting the sample through the capillary and through the aperture in the partition element.        
The total volume which, as a result of the application of negative pressure, is drawn from the measuring cell is continuously determined. The flow rate (μL/min) is determined from the volume which is drawn from the measuring cell per unit time.
The flow rate typically decreases over time, since the platelet plug gradually constricts the aperture and thus makes it difficult for the sample liquid to pass through.
The test result provided is the closure time in seconds. Closure time is defined as the time at which the flow rate was less than 10% of the initial flow rate for a period of three seconds. The initial flow rate is the flow rate at the start of the measurement when no platelet plug has yet formed at the aperture, but the dead volume has already been drawn from the system.
If the closure time of a patient's sample deviates from the reference range, this indicates a defect in thrombocyte function. Extended closure times indicate that there is thrombocyte dysfunction in terms of reduced aggregation ability. Shortened closure times indicate that there is thrombocyte dysfunction in terms of increased aggregation ability.
The closure time of a sample from a healthy donor with normal thrombocyte function depends on many factors.
Closure time is primarily affected by the type of measuring cell used. As explained above, measuring cells are used which contain different combinations of thrombocyte activators or thrombocyte inhibitors. When using a measuring cell containing collagen and epinephrine (Col/Epi), the closure time of a normal sample is between 84-160 seconds. When using a measuring cell containing collagen and ADP (Col/ADP), the closure time of a normal sample is between 68-121 seconds. When using a measuring cell containing ADP and prostaglandin E1 (ADP/PGE1), the closure time of a normal sample is below 106 seconds. The large variations in the reference ranges have the disadvantage that a closure time as such is not meaningful, but can only ever be interpreted in connection with the type of measuring cell used. A closure time of, for example, 130 seconds is a normal result for Col/Epi measuring cells, but an abnormal result for Col/ADP or ADP/PGE1 measuring cells in that it implies bleeding diathesis.
A further factor influencing closure time is the measuring cell architecture. Different diameters of the aperture or the capillary affect thrombus formation and hence the rate at which the aperture is closed.